Reflex testing of samples using residual materials from a prior test

ABSTRACT

Embodiments disclosed herein relate to methods and systems for performing automated assays, and particularly to performing sequential assays on a sample on an automated instrument.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/US2013/031072, filed Mar. 13, 2013, which claims priority toU.S. Provisional Application Ser. No. 61/624,198, entitled “REFLEXTESTING OF SAMPLES USING RESIDUAL MATERIALS FROM A PRIOR TEST,” filedApr. 13, 2012. Each of the above applications is hereby incorporated byreference in its entirety.

BACKGROUND

1. Technical Field

Embodiments disclosed herein relate to methods and systems forperforming sequential, automated assays, such as nucleic acid testingassays.

2. Description of the Related Art

Automation of molecular testing of samples has become increasinglycommon, due in part because automation can reduce the amount of timefrom sample collection to receiving results, can minimize experimentalvariability and can reduce the need for highly trained technicians. Inaddition to benefits in the field of diagnostics, automation ofprocessing and testing samples has facilitated high throughput testing.Automated devices for specimen and or sample processing typicallyinclude hardware and consumables. It is thus desirable to maximizeautomation of molecular testing, while minimize the amount ofconsumables used.

The embodiments described provide for improved automated specimen and/orsample testing that can advantageously be used in clinical and researchsettings.

SUMMARY

The present technology relates to methods and systems for performingsequential assays, such as nucleic acid testing assays. Some embodimentsrelate to sequential assays on one or more specimens performed on anautomated instrument. In some embodiments of the technology presentedherein, methods of performing an automated assay on a plurality ofsamples are provided that allow for improved reliability and ease of useassaying a plurality of specimens on an automated instrument. Themethods can include a) providing an automated instrument configured toreceive and process a plurality of samples from the plurality ofspecimens for one or more target analytes according to one or morerespective assay workflows; b) providing a plurality of samples to betested; c) automatically transferring a first portion of each of theplurality of samples to be tested to a respective plurality of firstvessels that comprise reagents for a first test for a first targetanalyte; d) automatically performing the first test on the portion ofthe plurality of samples to determine the presence of a first targetanalyte according to a first assay workflow; e) selecting a subsetsamples from the plurality of samples in which the presence of the firsttarget analyte was determined; f) automatically transferring a secondportion of the selected subset of samples from e) to a second vesselcomprising reagents for a second test to determine the presence of asecond target analyte according to a second assay workflow; and g)automatically performing the second test on the second portion of theselected subset of samples.

In some embodiments, each of the plurality of samples comprises apre-processed solution of isolated nucleic acids from a respectiveplurality of specimens. In some embodiments, the automated instrumentautomatically processes the plurality of specimens to obtain therespective plurality of samples comprising isolated nucleic acids.

In some embodiments, for each sample, the sample and the first vesselcomprising reagents for a first test are located on a first test strip,and such that step f) further comprises automatically transferring thesecond portion of the sample to the second vessel comprising reagentsfor the second test located on a second test strip.

In some embodiments, step e) further comprises e1) identifying a subsetof the plurality of first test strips for the second test; and e2) foreach first test strip in the subset, providing a corresponding secondtest strip comprising a vessel comprising reagents for a second test fora second analyte.

In some embodiments, the second test strip comprises at least onepipette tip. In some embodiments, the method comprises, prior to stepf), adding additional liquid to the sample.

In some embodiments, for each sample, the sample, the first vessel, anda receptacle for the second vessel are located on a single test strip.In some embodiments, method comprises, prior to step e), the steps ofd1) identifying a subset of the plurality of first test strips fortesting for the second analyte; and d2) for each test strip in thesubset, providing the second vessel comprising reagents for the secondtest in the receptacle.

In some embodiments, the method comprises, prior to step f), the step ofproviding a pipette tip configured to transfer the second portion of thesample to the second vessel comprising reagents for the second test.

In some embodiments, the pipette tip can be, for example, an unusedpipette tip or a washed pipette tip. In some embodiments, the methodcomprises, prior to step f), the step of adding additional liquid to thesample comprising.

In some embodiments, the first or the test comprises a reaction selectedfrom the group selected from: Polymerase Chain Reaction (PCR),Transcription Mediated Amplification (TMA), Oligonucleotide LigationAssay (OLA), Ligase Chain Reaction (LCR), Rolling Circle Amplification(RCA), Strand Displacement Amplification (SDA), and a hybridizationreaction.

In some embodiments, the method further comprises the step of comparingidentifying indicia on a test strip to a set of assay-specificidentifying data stored on the instrument.

Also presented herein is a system for performing an automated assay on aplurality of samples from a respective plurality of specimen, the systemcomprising an automated instrument configured to receive and process aplurality of samples according to one or more assay workflows; theinstrument comprising a plurality of test strips; a processor; a storagecapacity; and a program for performing an automated assay, the programcomprising instructions for a) providing an automated instrumentconfigured to receive and process a plurality of samples according toone or more assay workflows, the instrument comprising a plurality oftest strips; b) automatically transferring a first portion of eachsample to a respective plurality of first vessels comprising reagentsfor a first test; c) performing the first test to determine the presenceof a first target analyte; d) automatically transferring a secondportion of the samples, or a selected subset of the samples, torespective second vessels comprising reagents for a second test; and e)performing a second test on the second portion of the samples, orselected subset of samples, to determine the presence of a second targetanalyte.

In some embodiments of the above system, the program further comprisesinstructions for automatically isolating nucleic acid from the pluralityof specimens, to obtain a respective plurality of samples comprisingisolated nucleic acids.

In some embodiments of the above system, for each sample, the samplesand the first vessel comprising reagents for the first test are situatedon a first test strip, and wherein step e) comprises automaticallytransferring a portion of the extracted nucleic acid solution to asecond master mix tube situated on a second test strip.

In some embodiments of the above system, for each sample, the sample,the first vessel, and a receptacle for the second vessel are located ona single test strip, wherein the program comprises instructions for thesteps of d1) identifying a subset of the plurality of first test stripsfor testing for the second analyte; and d2) for each test strip in thesubset, providing a corresponding second vessel comprising reagents forthe second test in the receptacle.

Also presented herein is a method of performing reflex testing on aplurality of specimens, the method comprising a) providing a pluralityof specimens to be tested; b) processing the plurality of specimens toobtain a respective plurality of samples; c) transferring a firstportion of each of the plurality of samples to be tested to a respectiveplurality of first vessels that comprise reagents for a first test for afirst target analyte; d) performing the first test on the portion of theplurality of samples to determine the presence of a first targetanalyte; e) selecting a subset samples from the plurality of samples inwhich the presence of the first target analyte was determined for thereflex test; f) transferring a second portion of the selected subset ofsamples from e) to a second vessel comprising reagents for a second testto determine the presence of a second target analyte; and g) performingthe second test on the second portion of the selected subset of samples.

In some embodiments of the above methods, the first test comprises atest for the simultaneous detection of methicillin resistantStaphylococcus aureus and Staphylococcus aureus, and wherein the secondtest comprises a test for the detection of a determinant for mupirocinresistance. In some embodiments, the determinant for mupirocinresistance comprises the mupA gene.

Also presented herein is a method of performing reflex testing on aspecimen, the method comprising a) providing a specimen to be tested; b)processing the specimen to obtain a respective sample; c) transferring afirst portion of the sample to be tested to a first vessel comprisingreagents for a first test for a first target analyte; d) performing thefirst test on the first portion of the sample to determine the presenceof the first target analyte; f) transferring a second portion of thesample to a second vessel comprising reagents for a second test todetermine the presence of a second target analyte, if the first targetanalyte is detected in the first portion of the sample; and g)performing the second test on the second portion of the sample.

In some embodiments of the above method, the first test comprises a testfor the simultaneous detection of methicillin resistant Staphylococcusaureus and Staphylococcus aureus, and wherein the second test comprisesa test for the detection of a determinant for mupirocin resistance. Insome embodiments, the determinant for mupirocin resistance comprises themupA gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a test strip according to one embodiment.

FIG. 2 shows a test strip according to one embodiment.

FIG. 3 shows multiple test strips placed in a strip carrier according toone embodiment.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. All literature and similar materials cited in this applicationincluding, but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines oruses a term in such a way that it contradicts that term's definition inthis application, this application controls. While the present teachingsare described in conjunction with various embodiments, it is notintended that the present teachings be limited to such embodiments. Onthe contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

Automated diagnostic instruments capable of processing and testingmultiple specimens and/or samples in parallel have been described. Thesedevices can advantageously be used in high throughput to facilitate thespecimen and/or sample preparation and testing. By way of example,automated diagnostic instruments can prepare samples for diagnosticassays such as nucleic acid amplification assays, and performamplification and detection.

Diagnostic tests are, by nature, hypothesis-driven. When conducting ahypothesis driven test, such as testing a sample for a specific analyte,the result of the test can lead to a desire to test for another target,i.e., a reflex test. However, many times it is only useful to test asubset of the original samples, and would not be cost effective to testall of the samples. By way of example, in a clinical setting, it may bedesirable to test a specimen for one or more pathogens. If the testreveals the presence of a particular pathogen, it may be desirable toperform further testing, e.g., to determine the presence of antibioticresistance determinants. Obtaining an additional specimen for furthertesting, e.g., from a patient whose specimen tested positive for ananalyte indicative of the presence of a particular pathogen, can bedifficult and can delay further testing that is desirable. In additionto the potential difficulty in obtaining multiple specimens for testing,if multiple specimens are being tested, manual approaches to transferpatient specimens, or samples prepared from the specimens for testing(e.g., nucleic acid testing) to a new reaction vessel can be errorprone, as well as inefficient and labor intensive. Further, whenspecimens need to be processed prior to testing, it is desirable tominimize the costs associated with processing (e.g., reagents, and thelike). Thus, there exists a great need for improved methods to performreflex testing, in an automated way.

Accordingly, a “reflex test” refers to a subsequent test (e.g., a secondtest) that is undertaken based upon the results obtained in a previoustest (e.g., a first test). Non-limiting examples of first tests andreflex tests include, for example, a first test formethicillin-resistant Staphylococcus aureus MRSA andmethicillin-sensitive S. aureus (MSSA). For samples that test positivefor MRSA or MSSA, it may also be desirable to determine whether thesamples contain determinants for mupirocin (an antibiotic) resistance.As mupirocin resistance is typically relevant only for MRSA or MSSApositive samples, it is often not cost effective to test samplesnegative for MRSA and MSSA for mupirocin resistance. Various tests andreflex tests useful in the embodiments disclosed herein are discussed infurther detail below.

The methods and systems presented herein advantageously use residualmaterial from the original test rather than require a new specimen to beobtained from the patient. For example, it can be especiallyadvantageous to use residual material that has been highly processed andrequires little additional effort to conduct the reflex test.

Specimens and Samples

The embodiments disclosed herein can be used to test specimens usingautomated molecular assays. As used herein, the term “specimen” canrefer to a clinical specimen or sample from one or any number ofsources, including, but not limited to, bodily fluids (including, butnot limited to, blood, urine, serum, lymph, saliva, anal and vaginalsecretions, perspiration, peritoneal fluid, pleural fluid, effusions,ascites, purulent secretions, lavage fluids, drained fluids, brushcytology specimens, biopsy tissue, explanted medical devices, infectedcatheters, pus, biofilms and semen) of virtually any organism, withmammalian samples, particularly human samples, and environmental samples(including, but not limited to, air, agricultural, water and soilsamples) finding use in the invention. In addition, samples can be takenfrom food processing, which can include both input samples (e.g. grains,milk or animal carcasses), samples in intermediate steps of processing,as well as finished food ready for the consumer.

In some embodiments, samples are prepared from specimens, and tests areperformed on the samples. For example, in some embodiments, specimenscan be processed in order to obtain samples suitable for moleculartesting. In some embodiments, specimens can be analyzed directly, andare not pre-processed prior to testing. For example, a “direct sample”is a specimen that is collected from a subject and tested using themethods disclosed herein without isolating or culturing bacteria fromthe specimen, or without processing the specimen to isolate nucleicacids prior to testing. As such, direct samples are generally onlyminimally processed prior to screening. In some embodiments, thespecimens disclosed herein are processed to obtain samples suitable fortesting. For example, specimens containing cells can be processed tolyse cells and release the cellular components such as nucleic acids,proteins, and the like, prior to testing. In some embodiments, specimenscan be processed to produce samples that include isolated nucleic acids.As used herein, the phrase “isolate nucleic acids” refers to thepurification of nucleic acids from one or more cellular components. Theskilled artisan will appreciate that samples processed to “isolatenucleic acids” therefrom can include components and impurities otherthan nucleic acids. Samples that comprise isolated nucleic acids can beprepared from specimens using any acceptable method known in the art.For example, cells can be lysed using known lysis agents, and nucleicacids can be purified or partially purified from other cellularcomponents. Suitable reagents, and protocols for DNA and RNA extractionscan be found in, respectively, U.S. Patent Application Publication Nos.US 2010-0009351, and US 2009-0131650, each of which is incorporatedherein by reference in its entirety. In nucleic acid testing (e.g.,amplification and hybridization methods discussed in further detailbelow), he extracted nucleic acid solution can be added directly to areagents (e.g., either in liquid, bound to a substrate, in lyophilizedform, or the like, as discussed in further detail below), required toperform a test according to the embodiments disclosed herein.

In some embodiments, the tests, e.g., the first tests and the reflextests, described herein are tests to determine the presence of a targetanalyte in a specimen or sample. As used herein, the term “targetanalyte” can refer to various types of analytes of interest, includingfor example, target nucleic acids, target proteins, or other targetmolecules of interest. In some embodiments, the devices and methodsdescribed herein are used to perform reflex tests to determine thepresence of target nucleic acids, although the skilled artisan willappreciate that the embodiments disclosed herein can be readily adaptedto test for other types of target analytes.

In accordance with the above, the embodiments provided hereinadvantageously provide improved methods of automatically testingspecimens and samples for target analytes. The methods for performing anautomated assay on a plurality of samples. In some embodiments, themethod can a) providing an automated instrument configured to receiveand process a plurality of samples from said plurality of specimens forone or more target analytes according to one or more respective assayworkflows; b) providing a plurality of samples to be tested; c)automatically transferring a first portion of each of said plurality ofsamples to be tested to a respective plurality of first vessels thatcomprise reagents for a first test for a first target analyte; d)automatically performing the first test on the portion of said pluralityof samples to determine the presence of a first target analyte accordingto a first assay workflow; e) selecting a subset samples from theplurality of samples in which the presence of the first target analytewas determined; 0 automatically transferring a second portion of theselected subset of samples from e) to a second vessel comprisingreagents for a second test to determine the presence of a second targetanalyte according to a second assay workflow; and g) automaticallyperforming the second test on the second portion of the selected subsetof samples.

Automated instruments useful in the embodiments disclosed herein caninclude, for example, those described in U.S. Pat. No. 8,133,671, U.S.Patent Application Publication No. 2009-0111059, herein incorporated byreference in their entireties. The skilled artisan will appreciate thatthe embodiments disclosed herein can be readily adaptable to anysuitable automated system for specimen and/or sample processing andtesting, however. Desirably, the automated systems are configured toenable processing or testing of a plurality of samples according to oneor more workflows. As used herein, the terms “workflow,” “assayworkflow,” “assay,” “assay protocol,” “test,” and like terms refer to aprocedure for processing a specimen and/or sample. In typicalembodiments, a workflow can include sample preparation steps, such ascell lysis, nucleic acid extraction, nucleic acid purification, nucleicacid digestion, nucleic acid modification, protein extraction, proteinpurification, and the like. Several methods of nucleic acid extractionuseful in the embodiments disclosed herein are known in the art.Exemplary discussions of nucleic acid extraction can be found, forexample, in U.S. Patent Application Publication No. 2009-0131650, U.S.Patent Application Publication No. 2010-0009351, and U.S. PatentApplication Publication No. 2006-016623311/281,247, herein incorporatedby reference in their entireties. Likewise, exemplary discussions ofprotein extraction can be found, for example, in U.S. Pat. Nos.8,053,239 and 6,864,100, herein incorporated by reference in theirentireties.

In some typical embodiments, a workflow can also include nucleic acidamplification reactions. In some typical embodiments, a workflow canfurther include data analysis procedures.

As described above, in some embodiments, a portion (e.g., a firstportion, and, in some cases, a second portion) of the sample beingassayed or analyzed is transferred to a vessel that contains reagentsfor a test. As used herein, the term “vessel” refers to any type ofobject capable of holding a sample, including but not limited to, tubes,vessels in microtiter plates, and the like. In some embodiments, thevessel is located within a test strip. As used herein, the term “teststrip” or “reagent strip” refers to a package that holds one or moreconsumable components for an automated assay. The test strips cantherefore be configured for use by an apparatus that carries outautomated specimen and/or sample preparation, such an apparatus isdescribed, e.g., in International Patent Application Publication. No. WO09/054,870, incorporated herein by reference in its entirety.

In typical embodiments, a test strip can comprise a vessel, such as atube, well or the like, for holding a pre-processed sample, solutioncomprising isolated nucleic acids obtained from a specimen. Typically,the test strip can also comprise a vessel for holding reagents for amolecular test, such as a nucleic acid test (e.g., reagents used innucleic acid amplification, as discussed in further detail elsewhereherein). In some embodiments, the test strip can include additionalvessels for conducting sample preparation and for holding reagents andother consumables, such as pipette tips. In some embodiments, the teststrip or reagent strip includes one or more receptacles or aperturesthat are configured to receive reaction vessels that contain assayreagents. Accordingly, in some embodiments, vessels that containreagents for use in the assays described herein are unitary or integralwith the test strip, whereas in some embodiments, vessels that containreagents for use in the assays described herein are not integral withthe test strip, but are adapted to snap into a receptacle or aperture inthe test strip. Non-limiting examples of test strips or reagent stripsuseful in the embodiments disclosed herein are set forth in FIGS. 1 and2, in U.S. Pat. Nos. D618820, D637737, and in U.S. Provisional PatentApplication No. 61/541,991 filed Sep. 30, 2011 and entitled “UNITZEDREAGENT STRIP”, each of which is hereby incorporated by reference in itsentirety. FIG. 1 and FIG. 2 illustrate exemplary test strips/reagentstrips useful in the embodiments disclosed herein.

An automated instrument may comprise or be configured to receive, e.g.,in a dock, one or more strip carriers, each strip carrier or rackscapable of holding a plurality of test strips, and optionally tubes,e.g., tubes for carrying specimens. In typical embodiments, the teststrips are positioned in one or more strip carriers or racks that areaccommodated by the automated instrument. Exemplary strip carriers orracks useful in the embodiments disclosed herein are disclosed in, e.g.,U.S. Patent Application Publication No. 2009-0136386, although theskilled artisan will appreciate that various other configurations ofstrip holders or racks suitable for use with an automated system asdescribed herein can be used. In some embodiments, the test stripcarriers or racks are configured to hold a plurality of test strips orreagents strips, e.g., in various lanes. For example, the test stripcarrier can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or more, lanes, configured toaccommodate anywhere from 2-24 respective test strips or reagent strips.

In the methods disclosed herein, depending upon the results of the firsttest performed on the sample, a reflex test is performed. If resultsfrom the first test reveal the desirability of performing the reflex(subsequent) test, according to the methods disclosed herein, a second(or residual) portion of the sample left over following the first testprocedure, is automatically transferred a to a second vessel thatcomprises reagents for the reflex (subsequent test). The automatictransfer of the second portion of the sample can be performed in any oneof a number of methodologies. For example, in some embodiments, thevessel containing the remainder or residual portion of the samplefollowing the first test, can be transferred to a second test strip,after which, at least a portion of the sample is then automaticallytransferred to a vessel containing reagents for a second test. In someembodiments, the second portion of the sample is automaticallytransferred directly from the first test strip to a second master mixtube positioned in a second test strip. In some embodiments, the secondportion of the sample can be automatically transferred directly from thefirst test strip to a second vessel positioned the first test strip. Itwill be appreciated that although several embodiments are described ingreater detail below, any suitable methodology of automaticallytransferring the extracted nucleic acid solution to a second master mixtube can be performed according to the methods provided herein.

Transfer to a New Test Strip

In certain embodiments, for each sample, the sample and a vessel or tubecomprising reagents for the first test are situated on a single teststrip, e.g., a first test strip, and the step of automaticallytransferring a portion of the sample involves automatically transferringa second portion of the sample to a second master mix tube situated on asecond, discrete, test strip.

In some embodiments, the first and second test strips are situated inseparate strip carriers, or racks. As one non-limiting example, thefirst test strips can be positioned in lanes 1-6 of a first stripcarrier, and the second test strips can be positioned in lanes 1-6 of asecond strip carrier. An example of test strips positioned in a stripcarrier is shown in FIG. 3.

In some embodiments, the first and second test strips are situated inthe same strip carrier. As one non-limiting example, the first teststrips are positioned in lanes 1, 3, 5, 7, 9 and 11 and correspondingsecond test strips can be placed into lanes 2, 4, 6, 8, 10 and 12 of thesame strip carrier. As another non-limiting example, the first teststrips can be positioned in lanes 1-6 and the corresponding second teststrips can be positioned in lanes 7-12 of the same strip carrier. Itwill be appreciated that any suitable configuration of positioning forfirst and second test strips can be used. In some embodiments, theautomated instrument can use identifying indicia such as a barcode, RFIDcode, or the like, on each test strip to identify the position of thefirst and/or second test strip. In some embodiments, the method furthercomprises the step of comparing identifying indicia on a test, strip, avessel within a test strip, or a specimen tube, to a set ofassay-specific identifying data stored on the instrument.

Thus, in some embodiments, the user can select one or more first teststrips for a second, reflex test and transfer the one or more selectedtest strips into a new rack that contains second test strips, havingvessels comprising reagents for the second, reflex test. In someembodiments, the user can select one or more of the first test stripsfor a second, reflex test, and the automated instrument or user cantransfer a second portion of the sample to a second test strip locatedin a second, discrete test strip carrier or rack. In some embodiments,identifying indicia on specimen tubes, or test strips used in the firsttest can be accessed the automated instrument, and this information canbe used to track samples as they are processed during a second, orreflex test. Accordingly, in some embodiments of the above-describedmethods, the method can comprise, prior to transferring a second portionof the sample to a second vessel, identifying a subset of the pluralityof first test strips for further testing, e.g., for a second test; andfor each first test strip in the subset, providing a correspondingsecond test strip. The second test strip can include the second vesselcontaining reagents for the second test. In certain embodiments, thesecond test strip can also include at least one pipette tip.

Transfer to the Same Test Strip

In certain embodiments, for each sample, the sample solution and thefirst vessel that includes reagents for the first test are situated on afirst test strip, and the step of transferring a portion of theextracted nucleic acid solution comprises transferring a portion of theextracted nucleic acid solution to a second vessel containing reagentsfor a second test that is also located on the first test strip.

In some embodiments of the above-described methods, the method caninclude, prior to transferring a second portion of the sample to asecond vessel, the steps of identifying a subset of the plurality ofsamples for further testing, e.g., for a second or reflex test; and foreach of the samples identified for further testing, providing acorresponding second vessel including reagents for the second or reflextest on the first test strip. In a typical embodiment, the first teststrips are configured with an open position, e.g., a receptacle orvessel, suitable for adding an additional master mix tube to the strip.For example, the test strip can be a 4-snap strip, as illustrated inFIG. 2, which allows the user to add the reflex master mix to the openposition. In some embodiments, the test strips are arranged by the userin a new test strip holder or rack prior to performing the second orreflect test.

The skilled artisan will appreciate that it is desirable to minimize therisk of cross-contamination of reagents from the first test and reagentsfrom the second, or reflex, test. Accordingly, in some embodiments, theconsumables such as pipette tips can be replaced and/or cleaned usingresidual buffers in the test strip prior to conducting the reflex test.Thus, in certain embodiments, the method comprises, prior totransferring the second portion of the sample to the second vessel, thestep of providing a pipette tip. In some embodiments, the pipette tip isan unused pipette tip. In some embodiments, the pipette tip is a usedtip that has been washed to reduce or avoid contamination from previousliquid transfers. In certain embodiments, the method comprises, prior totransferring the second portion of the sample to the second vessel, thestep of washing a pipette tip that was used during the first test.

During the processing and/or testing of the samples prior to a second,reflex test, evaporation of the sample may occur. As such, sampleevaporation can reduce performance of a reflex test. The embodimentsdisclosed herein provide a solution to evaporation, by using a fixedvolume buffer, e.g., a wash buffer or nucleic acid elution buffer, toadd to the residual sample to ensure that there is sufficient volume tofurther process the residual sample, e.g., in a second, or reflex test.Accordingly, in some embodiments, the method comprises, prior toperforming a second or reflex test, the step of adding additional liquidto the residual sample. By way of example only, in some embodiments, theinitial volume of a sample can range from 1 μL-1 ml, and preferably isbetween 10 μL and 200 e.g., 25-75 μL. In some embodiments, a firstportion of about 5-25 μL, e.g, 10-20 μL of the sample, is removed forthe first test. By way of example, in some embodiments, 12.5 μL of theinitial sample is used in the first test. In order to ensure that asufficient volume of sample is available for the second test, in someembodiments, an additional volume of 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, 20 μL, 25μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75μL, 80 μL, 85 μL, 90 μL, 95 μL, 100 μL, 120 μL, 140 μL, 160 μL, 180 μL,200 μL, 300 μL, 400 μL, 500 μL, or more than 500 μL of additional liquidmay be added to increase the liquid volume prior to transfer of a secondportion of the sample to a vessel containing reagents for a second, orreflex test. Accordingly, the addition of solution to the remaining orresidual sample can compensate for evaporation that may occur before thesolution is transferred to the second master mix. In some embodiments, avolume equal to 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400% or more,relative to the volume of extracted nucleic acid solution remainingafter the first aliquot is transferred to the first master mix, may beadded to increase the liquid volume and compensate for evaporation thatmay occur before the solution is transferred to the vessel with reagentsfor a second test.

Nucleic Acid Testing (NAT) Assays and Reagents for NAT

As discussed above, the tests described herein can include, for examplenucleic acid testing. In some embodiments, the tests include testing fortarget nucleic acid sequences in a sample. Several forms of nucleic acidtesting are useful in the embodiments disclosed herein, including butnot limited to, testing that involves nucleic acid amplificationreactions. Several nucleic acid amplification reactions are known, andcan be used to determine the presence of target nucleic acids inaccordance with the embodiments disclosed herein. Methods of nucleicacid amplification can include, but are not limited to: polymerase chainreaction (PCR), strand displacement amplification (SDA), for examplemultiple displacement amplification (MDA), loop-mediated isothermalamplification (LAMP), ligase chain reaction (LCR), immuno-amplification,and a variety of transcription-based amplification procedures, includingtranscription-mediated amplification (TMA), nucleic acid sequence basedamplification (NASBA), self-sustained sequence replication (3SR), androlling circle amplification. See, e.g., Mullis, “Process forAmplifying, Detecting, and/or Cloning Nucleic Acid Sequences,” U.S. Pat.No. 4,683,195; Walker, “Strand Displacement Amplification,” U.S. Pat.No. 5,455,166; Dean et al, “Multiple displacement amplification,” U.S.Pat. No. 6,977,148; Notomi et al., “Process for Synthesizing NucleicAcid,” U.S. Pat. No. 6,410,278; Landegren et al. U.S. Pat. No. 4,988,617“Method of detecting a nucleotide change in nucleic acids”; Birkenmeyer,“Amplification of Target Nucleic Acids Using Gap Filling Ligase ChainReaction,” U.S. Pat. No. 5,427,930; Cashman, “Blocked-PolymerasePolynucleotide Immunoassay Method and Kit,” U.S. Pat. No. 5,849,478;Kacian et al., “Nucleic Acid Sequence Amplification Methods,” U.S. Pat.No. 5,399,491; Malek et al., “Enhanced Nucleic Acid AmplificationProcess,” U.S. Pat. No. 5,130,238; Lizardi et al., BioTechnology, 6:1197(1988); Lizardi et al., U.S. Pat. No. 5,854,033 “Rolling circlereplication reporter systems.” In some embodiments, two or more of thelisted nucleic acid amplification methods are performed, for examplesequentially.

As discussed above, in some embodiments, portions of a sample aretransferred to a vessel containing reagents for a test. For example, insome embodiments, the samples are transferred to a vessel that containsa “master mix” for a test or reaction, such as an amplification reactionor the like. The “master mix” can include some, or all of the componentsnecessary for a reaction, including, but not limited to enzymes,oligonucleotides, probes, salts, deoxynucleotide triphosphates, whichcomprises a polymerase enzyme and a plurality of nucleotides. In someembodiments, the master mix can further comprise hybridization probeswith detectable moieties, wherein the probes specifically hybridize totarget nucleic acids (and/or positive control target nucleic acidsequences). In some embodiments, a master mix can be provided at ahigher concentration than will be used in a reaction. In someembodiments, a master mix is provided in a lyophilized form, andreconstituted at a higher concentration that will be used in thereaction. In some embodiments a master mix includes reagents at aconcentration of at least about 2× of the reaction concentration, forexample 2×, 2.5×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 40×,50×, 100×, 200×, 250×, or 500×.

In some embodiments, the master mix can be in the form of one or morelyophilized pellets, as stored in a reagent tube on the test strip, andthe method can further include reconstituting the reagent pellet withliquid, e.g., a portion of a sample as described herein above to createa reaction/reagent mixture solution that is suitable for processing,e.g. testing.

In some embodiments, a “master mix” or can be provided in a vesselincludes identifying indicia, such as a bar code, RFID code, or thelike, that provides identifying information regarding the reagentscontained therein, e.g., indicia that identify that the vessel containsreagents for the detection of a particular target analyte.

Instruments and Systems

Also presented herein is a system for performing an automated assay, thesystem comprising an automated instrument configured to receive andprocess a plurality of specimens or samples according to one or moreassay workflows described herein above.

The system can also include a processor; a storage capacity; and aprogram for performing an automated assay. For example, the program caninclude instructions for an automated instrument configured to receiveand process a plurality of samples according to one or more assayworkflows as described herein. The program can include instructions forreceiving and processing a plurality of samples according to one or moreworkflows, wherein the samples are provided on a plurality of teststrips; automatically transferring a first portion of each sample to arespective plurality of first vessels comprising reagents for a firsttest; performing the first test to determine the presence of a firsttarget analyte; automatically transferring a second portion of thesamples, or a selected subset of the samples, to respective secondvessels comprising reagents for a second test; and performing a secondtest on the second portion of the samples, or selected subset ofsamples, to determine the presence of a second target analyte.

Automated instruments which can perform multiple assay protocolsconcurrently are known to those of skill in the art, and include but arenot limited to, BD MAX® (Becton Dickinson and Co., Franklin Lakes,N.J.), the VIPER® (Becton Dickinson and Co., Franklin Lakes, N.J.), theVIPER LT® (Becton Dickinson and Co., Franklin Lakes, N.J.), theSMARTCYCLER® (Cepheid, Sunnyvale, Calif.), ABI PRISM 7700® (AppliedBiosystems, Foster City, Calif.), ROTOR-GENE™ (Corbett Research, Sydney,Australia), LIGHTCYCLER® (Roche Diagnostics Corp, Indianapolis, Ind.),ICYCLER® (BioRad Laboratories, Hercules, Calif.), IMX4000® (Stratagene,La Jolla, Calif.), CFX96™ Real-Time PCR System (Bio-Rad LaboratoriesInc), and the like. Exemplary discussions of typical automatedinstruments for use with the methods provided herein can be found, forexample, in U.S. Pat. No. 8,133,671, which is incorporated herein byreference in its entirety.

It will be appreciated that the methods and systems described herein canapply to instruments that comprise 2, 3, 4 or more workstations whereinat least 2 of the workstations are supported by a common serviceresource. For example, an instrument with 4 workstations and a singlepipette head could still be compatibility controlled by the 2 indexconcept described herein.

As used herein, the terms storage capacity, storage device, storage andthe like can refer to any medium, device or means of storage ofinformation. Storage can include, but is not limited to, a disk drivedevice such as a hard drive, floppy disk, optical or magneto-opticaldisk, memory such as RAM or ROM chips, and any other medium used torecord or store data. In some embodiments, a storage capacity isconnected to a processor which sends information to be recorded on thestorage capacity after it is acquired. In specific embodiments, data isacquired by a system and is recorded on a storage capacity. In otherembodiments, data is acquired by a system and information is firstprocessed and the processed information is recorded on a storagecapacity.

The files and programs provided herein can be in any suitableprogramming language. In certain embodiments, the ADF utilizes XML as amechanism for formatting files. Further, in certain embodiments, ADFutilizes Python as a scripting language to provide a mechanism forexecuting result logic using common technologies available on theinstrument. It will be appreciated that any suitable file format andprogramming language can be utilized in the methods and systems providedherein. In certain embodiments, files can be encrypted to protectagainst the use of counterfeit reagents and to control specificparameter details on assay runs.

As used herein, an “input” can be, for example, data received from akeyboard, rollerball, mouse, voice recognition system or other devicecapable of transmitting information from a user to a computer. The inputdevice can also be a touch screen associated with the display, in whichcase the user responds to prompts on the display by touching the screen.The user may enter textual information through the input device such asthe keyboard or the touch-screen.

The embodiments disclosed herein are operational with numerous othergeneral purpose or special purpose computing system environments orconfigurations. Examples of well-known computing systems, environments,and/or configurations that may be suitable for use with the inventioninclude, but are not limited to, microcontrollers, personal computers,server computers, hand-held or laptop devices, multiprocessor systems,microprocessor-based systems, programmable consumer electronics, networkPCs, minicomputers, mainframe computers, distributed computingenvironments that include any of the above systems or devices.

As used herein, “instructions” refer to computer-implemented steps forprocessing information in the system. Instructions can be implemented insoftware, firmware or hardware and include any type of programmed stepundertaken by components of the system.

A “microprocessor” or “processor” may be any conventional generalpurpose single- or multi-core microprocessor such as a Pentium®processor, Intel® Core™, a 8051 processor, a MIPS® processor, or anALPHA® processor. In addition, the microprocessor may be anyconventional special purpose microprocessor such as a digital signalprocessor or a graphics processor. A “processor” may also refer to, butis not limited to, microcontrollers, field programmable gate arrays(FPGAs), application-specific integrated circuits (ASICs), complexprogrammable logic devices (CPLDs), programmable logic arrays (PLAs),microprocessors, or other similar processing devices.

The system is comprised of various modules as discussed in detailherein. As can be appreciated by one of ordinary skill in the art, eachof the modules comprises various sub-routines, procedures, definitionalstatements and macros. Each of the modules are typically separatelycompiled and linked into a single executable program. Therefore, thefollowing description of each of the modules is used for convenience todescribe the functionality of the preferred system. Thus, the processesthat are undergone by each of the modules may be arbitrarilyredistributed to one of the other modules, combined together in a singlemodule, or made available in, for example, a shareable dynamic linklibrary.

Certain embodiments of the system may be used in connection with variousoperating systems such as SNOW LEOPARD®, iOS®, LINUX, UNIX or MICROSOFTWINDOWS®.

Certain embodiments of the system may be written in any conventionalprogramming language such as assembly, C, C++, BASIC, Pascal, or Java,and run under a conventional operating system.

In addition, the modules or instructions may be stored onto one or moreprogrammable storage devices, such as FLASH drives, CD-ROMs, hard disks,and DVDs. One embodiment includes a programmable storage device havinginstructions stored thereon.

In some embodiments of the above system, the system further can comprisea device for reading identifying indicia on reagent packaging, e.g., ontest strips, vessels containing reagents, as well as identifyingindicial present on vessels that contain samples or specimens. It willbe appreciated that any suitable device for reading identifying indiciacan be used in the systems provided herein. Likewise, any suitableidentifying indicia may be used that is compatible with the device onthe instrument. Examples include bar codes, QR codes, RFID tags, colorcodes and the like. In typical embodiments, the device can be a bar codereader, and the identifying indicia can comprise a bar code.

Exemplary Reflex Assays

By way of another example, after a positive MRSA assay, a reflex assayfor vancomycin resistance determinant(s), Panton-Valentine leukocidin(PVL) determinants, or to classify MRSA by spa-typing, or testingstaphylococcal cassette chromosome mec (SCCmec) typing, can be performedfor specimen that test positive for MRSA. Suitable tests for vancomycinresistance determinant(s), Panton-Valentine leukocidin (PVL)determinants, or to classify MRSA by spa-typing, or testing SCCmec areknown in the art, as exemplified by Mak et al, J. Clin. Microbiol.(2009) 12:4136; Reischl et al. Eur. J. Clin. Microbiol. Infect. Dis.(2007) 26:131-135; Narukawa et al., Tohoku J. Exp. Med. (2009)218:207-213; Chongtrakool et al., Antimicrob. Agents Chemother. (2006)50:1001-1012; each of which is incorporated by reference in itsentirety.

By way of another example, using a stool specimen from patients withsevere diarrhea, first tested for Clostridium difficile, a reflex assaycould be performed on positives to determine toxin subtype, or onnegatives to test for a different causative organism. The converse isalso true where stool specimens that are tested for an enteric panelcould be reflexed to a Clostridium difficile test. Suitable tests forClostridium difficile, and for toxin subtypes are known in the art, asexemplified by Kvach et al. J. Clin. Microbiol. (2010) 48:109-114; andNorthey et al., J. Med. Microbio. (2005) 54:543-547; each of which isincorporated by reference in its entirety.

By way of another example, using a cerebro-spinal fluid specimen frompatients with high fever, the specimens or samples can be first testedfor a viral infection using a total nucleic acid test, and a second testcan be performed on negatives to look for bacterial targets. Suitabletests for viral and bacterial infections are known in the art, asexemplified by Mahoney et al, J. Clin. Microbiol. (2007) 45:2965-2970and Melendez et al. Clin. Microbiol. Infect. (2010) 16:1762-1769; eachof which is incorporated by reference in its entirety.

By way of another example, using sputum specimens, the specimens orsamples could be first tested for Mycobacterium tuberculosis complex(MTBC) and rifampin resistance. If there are positives, a reflex couldbe performed for Isoniazid and fluoroquinolone resistance in order todetermine if the strain is MDR or XDR. Suitable tests for Mtbc,rifampin, isoniazid and fluoroquinolone resistance are known in the art,as exemplified by Somoskovi et al. J. Clin. Microbiol. (2003)41:2822-2826; Saribas et al., J. Clin. Microbiol. (2003) 41:816-818;Rindi et al. J. Microbiol. Methods (2003) 55:797-800; Ip et al., J.Clin. Microbiol. (2006) 4:970-975; each of which is incorporated byreference in its entirety.

By way of another example, using bronchoalveloar aspirates in ICUpatients, an initial screening for KPC/OXA/NDM carbapenemases can beperformed. If negatives are found, a VIM/IMP screening could beperformed. Suitable tests KPC/OXA/NDM detection and for VIM/IMPscreening are known in the art, as exemplified by Nordmann et al. Clin.Microbiol. Infect. (2002) 8:321-331; and Monteiro et al. J. Antimicrob.Chemother. (2012); each of which is incorporated by reference in itsentirety.

By way of another example, using a sample, and following a Gram stain ora maldi identification, an assay could be performed on a panel ofresistance markers panel for Gram positive (if the Gram is positive) ora Gram negative panel (if the Gram is negative). Here the reflexstrategy described is after another system or test for identification.Suitable tests for Gram positive and Gram negative screening are knownin the art, as exemplified by Carroll et al. J. Clin. Microbiol. (2000)5:1753-1757; which is incorporated by reference in its entirety.

By way of another example, using food samples or environmental samples,a Listeria spp. screening can be first performed. If positives areidentified, a specific reflex assay for L. monocytogenes can beperformed. Suitable tests detecting Listeria spp and L. monocytogenesare known in the art, as exemplified by Bubert A. App. Environ.Microbiol. (1999) 10:4688-4692; and Borucki et al., J. Clin. Microbiol.(2003) 41:5537-5540; each of which is incorporated by reference in itsentirety.

By way of another example, using water samples or pharmaceuticalenvironment samples, a TVC assay can be performed. If the initial assayis positive, a reflex assay can be performed for Gram positive or Gramnegative or a specific assay towards E. coli or enterococci or bacillus.Suitable tests for TVC and for Gram positive and Gram negative screeningare known in the art, as exemplified by Ereveeval et al., J. Clin.Microbiol. (2003) 41:5466-5472; and Carroll et al. J. Clin. Microbiol.(2000) 5:1753-1757; each of which is incorporated by reference in itsentirety.

Another exemplary embodiment relates to the testing of specimens fromsubjects having, or suspected of having cystic fibrosis. For example, afirst test can be performed on samples or specimens from a subject totest for the presence of target analytes, e.g., target nucleic acidsequences, associated with Psuedomonas aeruginosa, and/or Burkholderiacepacia. Non-limiting examples of assays for the detection of P.aeruginosa and/or B. cepacia useful in the embodiments disclosed hereininclude, e.g., those described in Spilker et al. (2004), J. Clin.Microbiol. 42(5):2074-2079, and Vonberg et al., (2006), J. Med.Micriobiol. 55(Pt. 6):721-727. For samples testing positive for eitherpathogen, reflex tests for antibiotic resistance determinants can beperformed, e.g., tests to determine target nucleic acids indicative oftetracycline, nalidixic acid, norfloacin, chloramphenicol, cirpfloxacin,or the like.

It will be appreciated that the above list is meant only to provideexamples of the methods and systems provided herein, and that anysuitable reflex assay could be performed based upon the outcome of aninitial assay.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting.

Example 1 Identification of Samples for Reflex Analysis

This example describes the identification of samples for reflex analysisat the end of a PCR run using an automated instrument for samplepreparation, processing and analysis. A panel of 12 biologicalspecimens, each in an individual sample tube, is processed to obtainrespective samples of solution comprising isolated nucleic acids. Eachtest strip includes a tube for cell lysis, indicated as “Reaction Tube”in FIG. 1. The test strip also includes positions for three additionaltubes, indicated as positions 1, 2 and 3 in FIG. 1. At position 3 is aconical tube configured for holding the final extracted nucleic acidsolution, or sample solution that includes isolated nucleic acids. Asshown in FIG. 1, the test strip also comprises additional reservoirpositions in the test strip to hold extraction solutions, a wastechamber to hold waste liquid, and several sheaths to hold disposablepipettes. The test strip is also configured with identifying indiciasuch as a bar code at one end of the strip.

The user places an extraction tube in position 1 and a master mix fordetection and/or identification of methicillin-resistant Staphylococcusaureus (MRSA) and S. aureus (including methicillin sensitive S. aureus),e.g., the BD GENEOHMT™ StaphSR™ assay reagents (Becton Dickinson,Franklin Lakes, N.J.), in position 2 of each of the 12 test strips. Eachspecimen is automatically processed with lysis buffer in the reactiontube, and a portion of the resulting liquid is automatically transferredto the extraction tube, where bead-based nucleic acid extraction isperformed using the extraction solutions and the final extracted nucleicacid solution sample is transferred to the conical tube at position 3.The total volume of the extracted nucleic acid solution sample in theconical tube of each test strip is 25 μL. An aliquot of 12.5 μL of theextracted nucleic acid solution is automatically pipetted to theadjacent master mix tube to reconstitute the StaphSR™ master mix. Themaster mix is transferred to a reaction cartridge for PCR analysis.After PCR, 6 of the 12 samples are indicated as positive for MRSA or S.aureus, and identified as candidates for mupirocin resistance, and aretherefore selected for further analysis as set forth in the examplesbelow.

Example 2 Manual Transfer of Extracted Nucleic Acid Solution to New TestStrip and Automatic Transfer to New Master Mix Tube for Reflex PCRANALYSIS

Sample preparation and processing using PCR analysis for MRSA and S.aureus are performed as described above in Example 1. Each of the 6 usedtest strips corresponding to the samples identified for reflex analysisfor mupirocin resistance are repositioned in lanes 1-6 of a new rack.New test strips are positioned in lanes 1-6 of a separate rack. Anadditional 15 μL of elution buffer is added to the remaining extractednucleic acid solution in each test strip associated with positivesamples from the StaphSR™ test performed in Example 1, above. A portionof the residual or remaining sample comprising isolated nucleic acidsobtained from the specimen is transferred to position 3 of a new teststrip. The user places a master mix for mupirocin resistance PCRanalysis in position 2 of each of the 6 new test strips. An aliquot of12.5 μL of the extracted nucleic acid solution is automatically pipettedto the adjacent master mix tube to reconstitute the master mix for themupirocin resistance PCR analysis, using a new pipette situated on thenew test strip.

The reflex PCR analysis indicates that a subset of the samples selectedfor the reflex test are positive for mupirocin resistance.

Example 3 Automatic Transfer of Extracted Nucleic Acid Solution to NewMaster Mix Tube on New Test Strip for Reflex PCR Analysis

Sample processing and testing using the StaphSR™ assay are performed asdescribed above in Example 1. Each of the 6 used test stripscorresponding to the samples identified for reflex analysis formupirocin resistance are repositioned in lanes 1-6 of a new rack. Newtest strips are positioned in lanes 1-6 of a separate rack. Anadditional 15 μl, of elution buffer is added to the remaining extractednucleic acid solution in each test strip in lanes 1-6. An aliquot of12.5 μL of the extracted nucleic acid solution is automatically pipettedto a master mix tube on the corresponding new test strip to reconstitutethe master mix for the mupirocin resistance PCR analysis, using a newpipette situated on the new test strip.

The reflex PCR analysis indicates that a subset of the samples selectedfor the reflex test are positive for mupirocin resistance.

Example 4 Automatic Transfer of Extracted Nucleic Acid Solution to NewMaster Mix Tube on the Same Test Strip for Reflex PCR Analysis

Sample processing and PCR analysis for MRSA are performed as describedabove in Example 1, with the exception that a 4-snap strip is used asshown in FIG. 2. Specifically, the 4-snap strip is configured in asimilar fashion to the test strip used in Examples 1-3, but the stripcomprises one additional position (“position 4”) for optional placementof an additional master mix tube. For each of the 6 samples identifiedfor reflex analysis for mupirocin resistance, an additional 15 μL ofelution buffer is added to the remaining extracted nucleic acid solutionin each test strip. The user places a master mix for mupirocinresistance PCR analysis in position 4 of each of the 6 test stripsidentified for reflex analysis. An aliquot of 12.5 μL of the extractednucleic acid solution is automatically pipetted to the master mix tubeat position 4 to reconstitute the master mix for the mupirocinresistance PCR analysis, using a new pipette situated on the new teststrip. Nucleic acid amplification is performed as described above.

The reflex PCR analysis indicates that a subset of the samples selectedfor the reflex test are positive for mupirocin resistance.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the embodiments belong. Although any methods andmaterials similar or equivalent to those described herein may also beused in the practice or testing of the embodiments, the preferredmethods and materials are now described.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amethod” includes a plurality of such methods and equivalents thereofknown to those skilled in the art, and so forth.

All references cited herein including, but not limited to, published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

The steps of a method or algorithm described in connection with theembodiments disclosed herein may be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module may reside in RAM memory, flash memory, ROM memory,EPROM memory, EEPROM memory, registers, hard disk, a removable disk, aCD-ROM, or any other form of storage medium known in the art. Anexemplary storage medium may be coupled to the processor such theprocessor can read information from, and write information to, thestorage medium. In the alternative, the storage medium may be integralto the processor. The processor and the storage medium may reside in anASIC. The ASIC may reside in a user terminal. In the alternative, theprocessor and the storage medium may reside as discrete components in auser terminal.

What is claimed is:
 1. A method of performing an automated assay on a plurality of specimens, said method comprising: a) providing an automated instrument configured to receive and process a plurality of samples from said plurality of specimens for one or more target analytes according to one or more respective assay workflows; b) providing a plurality of samples to be tested; c) automatically transferring a first portion of each of said plurality of samples to be tested to a respective plurality of first vessels that comprise reagents for a first test for a first target analyte; d) automatically performing the first test on the portion of said plurality of samples to determine the presence of a first target analyte according to a first assay workflow; e) selecting a subset samples from the plurality of samples in which the presence of the first target analyte was determined; f) automatically transferring a second portion of the selected subset of samples from e) to a second vessel comprising reagents for a second test to determine the presence of a second target analyte according to a second assay workflow; and g) automatically performing the second test on the second portion of the selected subset of samples.
 2. The method of claim 1, wherein each of the plurality of samples comprises a pre-processed solution of isolated nucleic acids from a respective plurality of specimens.
 3. The method of claim 2, wherein the automated instrument automatically processes the plurality of specimens to obtain the respective plurality of samples comprising isolated nucleic acids.
 4. The method of claim 1, wherein, for each sample, said sample and said first vessel comprising reagents for a first test are located on a first test strip, and wherein step f) further comprises automatically transferring the second portion of the sample to the second vessel comprising reagents for the second test located on a second test strip.
 5. The method of claim 4, wherein step e) further comprises e1) identifying a subset of said plurality of first test strips for the second test; and e2) for each first test strip in said subset, providing a corresponding second test strip comprising a vessel comprising reagents for a second test for a second analyte.
 6. The method of claim 4, wherein said second test strip comprises at least one pipette tip.
 7. The method of claim 4, wherein said method comprises, prior to step f), adding additional liquid to said sample.
 8. The method of claim 1, wherein, for each sample, said sample, said first vessel, and a receptacle for said second vessel are located on a single test strip.
 9. The method of claim 8, wherein said method comprises, prior to step e), the steps of: d1) identifying a subset of said plurality of first test strips for testing for the second analyte; and d2) for each test strip in said subset, providing the second vessel comprising reagents for the second test in the receptacle.
 10. The method of claim 8, wherein said method comprises, prior to step f), the step of providing a pipette tip configured to transfer said second portion of the sample to the second vessel comprising reagents for the second test.
 11. The method claim 10, wherein said pipette tip is an unused pipette tip.
 12. The method claim 10, wherein said pipette tip is a washed pipette tip.
 13. The method of claim 8, wherein said method comprises, prior to step f), the step of adding additional liquid to said sample comprising.
 14. The method of claim 1, wherein said first or said test comprises a reaction selected from the group selected from: Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Oligonucleotide Ligation Assay (OLA), Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and a hybridization reaction.
 15. The method of claim 1, wherein said method further comprises the step of comparing identifying indicia on a test strip to a set of assay-specific identifying data stored on the instrument.
 16. The method of claim 1, wherein the first test comprises a test for the simultaneous detection of methicillin resistant Staphylococcus aureus and Staphylococcus aureus, and wherein the second test comprises a test for the detection of a determinant for mupirocin resistance.
 17. The method of claim 16, wherein the determinant for mupirocin resistance comprises the mupA gene.
 18. A system for performing an automated assay on a plurality of samples from a respective plurality of specimen, said system comprising: an automated instrument configured to receive and process a plurality of samples according to one or more assay workflows; said instrument comprising a plurality of test strips; a processor; a storage capacity; and a program for performing an automated assay, said program comprising instructions for: a) providing an automated instrument configured to receive and process a plurality of samples according to one or more assay workflows, said instrument comprising a plurality of test strips; b) automatically transferring a first portion of each sample to a respective plurality of first vessels comprising reagents for a first test; c) performing the first test to determine the presence of a first target analyte; d) automatically transferring a second portion of the samples, or a selected subset of the samples, to respective second vessels comprising reagents for a second test; and e) performing a second test on the second portion of the samples, or selected subset of samples, to determine the presence of a second target analyte.
 19. The system of claim 18, wherein the program further comprises instructions for automatically isolating nucleic acid from the plurality of specimens, to obtain a respective plurality of samples comprising isolated nucleic acids.
 20. The system of claim 18, wherein, for each sample, said samples and said first vessel comprising reagents for the first test are situated on a first test strip, and wherein step e) comprises automatically transferring a portion of said extracted nucleic acid solution to a second master mix tube situated on a second test strip.
 21. The system of claim 18, wherein, for each sample, said sample, said first vessel, and a receptacle for said second vessel are located on a single test strip, wherein said program comprises instructions for the steps of: d1) identifying a subset of said plurality of first test strips for testing for the second analyte; and d2) for each test strip in said subset, providing a corresponding second vessel comprising reagents for the second test in the receptacle.
 22. A method of performing reflex testing on a plurality of specimens, said method comprising: a) providing a plurality of specimens to be tested; b) processing the plurality of specimens to obtain a respective plurality of samples; c) transferring a first portion of each of said plurality of samples to be tested to a respective plurality of first vessels that comprise reagents for a first test for a first target analyte; d) performing the first test on the portion of said plurality of samples to determine the presence of a first target analyte; e) selecting a subset samples from the plurality of samples in which the presence of the first target analyte was determined for the reflex test; f) transferring a second portion of the selected subset of samples from e) to a second vessel comprising reagents for a second test to determine the presence of a second target analyte; and g) performing the second test on the second portion of the selected subset of samples.
 23. A method of performing reflex testing on a specimen, said method comprising: a) providing a specimen to be tested; b) processing said specimen to obtain a respective sample; c) transferring a first portion of said sample to be tested to a first vessel comprising reagents for a first test for a first target analyte; d) performing said first test on said first portion of said sample to determine the presence of said first target analyte; f) transferring a second portion of said sample to a second vessel comprising reagents for a second test to determine the presence of a second target analyte, if said first target analyte is detected in said first portion of said sample; and g) performing said second test on said second portion of said sample.
 24. The method of claim 23, wherein the first test comprises a test for the simultaneous detection of methicillin resistant Staphylococcus aureus and Staphylococcus aureus, and wherein the second test comprises a test for the detection of a determinant for mupirocin resistance.
 25. The method of claim 24, wherein the determinant for mupirocin resistance comprises the mupA gene. 